Polyvalent anti-tumor fibroblast vaccine

ABSTRACT

Compositions of matter, of production, and treatment modalities are disclosed for the prevention and/or therapeutic reduction of tumors through induction of immunity against tumor associated fibroblasts and components of tumor microenvironment. In one embodiment of the invention, placentally derived fibroblast cells are cultured under conditions replicating tumor microenvironment. Expression of CD248 on said cultured fibroblasts is used as a marker of effective manipulation. Cells expressing CD248 are utilized a immunogens for stimulation of immunity towards cancer associated fibroblastic cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 14/992,972 filed on Jan. 11, 2016 which claims the benefit of U.S. Provisional Application No. 62/101,444 filed on Jan. 9, 2015, the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention pertains to the field of tumor immunotherapy, more specifically to the field of cancer vaccination. The invention relates to the area of utilizing the immune response to selectively attack tumor associated stromal tissue, specifically, “activated fibroblasts” which have been influenced by the tumor but are not neoplastic themselves.

BACKGROUND OF THE INVENTION

Growth of various types of cancers is associated with a number of characteristic cellular and molecular changes in the surrounding stroma cells. One highly consistent feature of the reactive stroma of numerous types of epithelial cell cancer is the induction of the fibroblast activating protein alpha (from now on referred to as FAP-alpha or FAP), a cell surface molecule of the reactive stromal fibroblast which was originally identified with the monoclonal antibody F19. Since the FAP is selectively expressed in stroma of a number of epithelial cell carcinomas, irrespective of the site and histological type of the carcinoma, it was desirable to develop a treatment concept for the FAP-alpha target molecule in order to allow imaging techniques, the diagnosis and treatment of epithelial cell cancer and many other syndromes. For this purpose a monoclonal murine antibody named F19 was developed which specifically binds to FAP. This antibody was described in U.S. Pat. No. 5,059,523 and WO 93/05804 which are included in their entirety in this document by reference. Unfortunately agents that actually reduce or kill tumor associated stroma do not exist.

There is evidence that an immune response can effectively reduce tumors. Studies of adoptive T cell immunotherapy [1] along with recently reported positive clinical results in non-Hodgkins lymphoma [2, 3] and prostate cancer immunotherapy targeting tumor-associated antigens (TAAs) have provided proof of concept that the immune system can support a clinically effective anti-tumor immune response.

Although the benefits of anti-tumor immunotherapy has not been demonstrated in a wide range of tumor types, it has been postulated that the missing critical element is a sufficiently potent, readily translatable cancer vaccine strategy [4]. Patients with cancer can have endogenous or immunotherapy elicited humoral and cellular responses to several tumor-associated antigens (TAAs) [5-14], however, these have generally been of sub-optimal magnitude with elusive clinical efficacy. Additionally, cancer patients with significant inflammatory infiltrates, i.e. medullary carcinoma, have significantly improved survival despite greater cellular anaplasia [15-17]. Thus, it is reasonable to hypothesize that a sufficiently potent, antigen-specific immunotherapeutic strategy for cancer would have clinical efficacy and offer a valuable new treatment modality.

A variety of TAAs have been identified in cancer consisting of overexpressed normal proteins and mutated proteins that are normally found in tissue, however, only a minority of the TAAs that have been discovered so far are immunogenic, which limits the potential use for immunotherapy. In addition, while the overwhelming majority of TAAs are expressed in tumor cells, they are typically also expressed in a variety of normal cells, e.g. the cancer TAAs; epidermal growth factor receptors (HER2), carcinoembryonic antigen (CEA), mucin (MUC1), the tumor suppressor protein p53, and telomerase reverse transcriptase (TERT). Thus, they are recognized by the immune system as self-molecules, and the immune system has protective mechanisms for preventing recognition of self-tissue antigens and autoimmune responses. Additionally, tumors employ other mechanisms for escaping immune surveillance, such as: (i) low level expression of MHC class I molecules [18]; (ii) lack of expression of B7 (CD80/CD86) co-stimulatory molecules [19]; (iii) production of cytokines that stimulate the accumulation of immune-suppressor cells [20, 21]; and (iv) ineffective processing and presentation of self-antigens by “professional” antigen-presenting cells (APC) [22]. This probably explains why TAA or tumor cell vaccines that have been used in clinical trials generally do not induce strong protective immunity [1].

To address why some of the clinical vaccines have not worked as potently as one would have desired, t is important to look to the basic research experiments conducted in this area. The era of molecular biology has allowed for gene-specific deletion in animals. This means that genes associated with immune responses can be “knocked-out” of animals so has to study the importance of the specific gene in an in vivo setting. Speaking in very general terms, there are two pathways that the immune system can take when it is activated. The first type is called “Th1”, which is involved in destroying cells of the body that are infected from the inside, such as virally infected cells. The second type of immune response is called “Th2”, which is responsible for killing targets that reside outside of the cells of the body [23, 24]. Since cancer consists of cells of the body that have distinguishing properties from the other cells (ie high proliferation, ability to metastasize, etc), it would make sense that the Th1 path of the immune response would be the one responsible for control of cancer, if the immune response is involved at all. Indeed, Coley's toxin (and its constituents) were identified decades later to be potent inducers of the Th1 cytokine TNF-alpha, as well as activators of this general immune response pathway [25, 26]. The discovery of sine qua non transcription factors for Th1 and Th2 immunity allowed the hypothesis to be tested in animal models if these pathways play an role in control of cancer. Transcription factors for Th1 immunity are T-bet [27], and STAT4 [28], and for Th2 are GATA-3 [29], STAT6 [30, 31]. When various tumors are administered to STAT6 knockout animals (therefore having a Th1 predisposition since the Th2 pathway is ablated), these tumors are either spontaneously rejected [32], or immunity to them is achieved with much higher potency compared to wild-type animals [33]. Furthermore, in STAT6 knockout animals, immunologically mediated resistance to metastasis formation is observed [34]. On the other hand, the STAT4 knockout mice which lack Th1 capability and therefore have upregulated Th2 immunity, accelerated cancer formation occurs after treatment with chemical carcinogens [35].

While the above suggests an importance of the Th1 immune response in controlling tumors, in many cases animal data does not translate efficiently to the clinical situations. Accordingly, we turn our attention to situations where immune suppression is induced either by genetic abnormality or in response to a medical condition. Generally speaking, natural killer (NK) cell activity is associated with Th1 immune responses and tumor immunity [36]. Patients with the congenital abnormality Chediak-Higashi Syndrome, are characterized by absent or severely diminished NK function. In this population, the overall incidence of malignant tumors is 200-300 times greater than that in the general population [37]. Another example of an in-born trait associated with immune deviation is patients born with a specific polymorphism of the IL-4 receptor gene that is known to be associated with augmented Th2 responses. Multivariate regression analysis showed that this polymorphism was an independent prognostic factor for shorter cancer survival and more advanced histopathological grade [38]. In addition to inborn genetic abnormalities, the immune suppressive regimens used for post-transplant antirejection effect are associated with a selective inhibition of Th1 responses [39-41]. In support of the concept that suppression of Th1 immunity is associated with cancer onset, the incidence of cancer in the post-transplant population is markedly increased in comparison to controls living under similar environmental conditions [42-47]. In terms of disease associated immune suppression, HIV infected patients also have a marked predisposition to a variety of tumors, especially, but not limited to lymphomas, as a result of immunodeficiency [48].

Although the above examples support a relation between immune suppression (or Th2 deviation) and cancer, the opposite situation, of immune stimulation resulting in anticancer response is also documented. Numerous clinical trials using antigen specific approaches such as vaccination with either tumor antigens alone [49, 50], tumor antigens bound to immunogens [51, 52], tumor antigens delivered alone [53] or in combination with costimulatory molecules by viral methods [54], tumor antigens loaded on dendritic cells ex vivo [55-57], or administration of in vitro generated tumor-reactive T cells [58], have all demonstrated some, albeit modest clinical effects. Furthermore, the influence of the immune system on cancer does not necessarily have to lead to regression. It is documented that inappropriate immune responses (broadly speaking Th2 responses) can actually stimulate tumor growth [59, 60]. Accordingly, the previous mentioned evidence all support the recognition of cancer by the immune system, and the notion that the immune system, if stimulated properly may lead to cancer regression.

The immune system is not responsible for seeing only “self” versus “non-self” but actually seeing an responding to different variations of “self”. The tumor, in its quest for proliferative advantage, ability to metastasize, and need for formation of new blood supply, actually expresses new molecules at levels that are recognized by the immune system. Immunological recognition of molecules needed for the tumor to have the “cancer phenotype” has been well-documented. We will not overview all of these here but provide some examples. Specifically, the proliferative advantage of tumors is associated with growth factor receptor upregulation, accordingly immune responses to various such receptors are known to exist naturally, or to be inducible [61, 62]. The same is true for matrix metalloproteases involved in tumor extravasation and metastasis [63, 64], as well as for angiogenic factors involved in formation of new blood vessels [65, 66]. The invention seeks to utilize immunological targeting of the tumor associated fibroblasts as a means of blocking tumor growth and metastasis.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of advancing and clarifying the principles of the invention disclosed herein, reference will be made to certain embodiments and specific language will be used to describe said embodiments. It will nevertheless be understood and made clear that no limitation of the scope of the invention is thereby intended. The alterations, further modifications and applications of the principles of the invention as described herein serve only as specific embodiment, however one skilled in the art to which the invention relates will understand that the following are indeed only specific embodiments for illustrative purposes, and will derive similar types of applications upon reading and understanding this disclosure.

To allow for the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

“antigen-presenting cells” or “APCs” are used to refer to autologous cells that express MHC Class I and/or Class II molecules that present antigens to T cells. Examples of antigen-presenting cells include, e.g., professional or non-professional antigen processing and presenting cells. Examples of professional APCs include, e.g., B cells, whole spleen cells, monocytes, macrophages, dendritic cells, fibroblasts or non-fractionated peripheral blood mononuclear cells (PMBC). Examples of hematopoietic APCs include dendritic cells, B cells and macrophages. Of course, it is understood that one of skill in the art will recognize that other antigen-presenting cells may be useful in the invention and that the invention is not limited to the exemplary cell types described herein. APCs may be “loaded” with an antigen that is pulsed, or loaded, with antigenic peptide or recombinant peptide derived from one or more antigens. In one embodiment, a peptide is the antigen and is generally antigenic fragment capable of inducing an immune response that is characterized by the activation of helper T cells, cytolytic T lymphocytes (cytolytic T cells or CTLs) that are directed against a malignancy or infection by a mammal. In one, embodiment the peptide includes one or more peptide fragments of an antigen that are presented by class I MHC or class II MHC molecules. The skilled artisan will recognize that peptides or protein fragments that are one or more fragments of other antigens may used with the present invention and that the invention is not limited to the exemplary peptides, tumor cells, cell clones, cell lines, cell supernatants, cell membranes, and/or antigens that are described herein.

“dendritic cell” or “DC” refer to all DCs useful in the present invention, that is, DC is various stages of differentiation, maturation and/or activation. In one embodiment of the present invention, the dendritic cells and responding T cells are derived from healthy volunteers. In another embodiment, the dendritic cells and T cells are derived from patients with cancer or other forms of tumor disease. In yet another embodiment, dendritic cells are used for either autologous or allogeneic application.

“effective amount” refers to a quantity of an, antigen or epitope that is sufficient to induce or amplify an immune response against a tumor antigen, e.g., a tumor cell.

“vaccine” refers to compositions that affect the course of the disease by causing an effect on cells of the adaptive immune response, namely, B cells and/or T cells. The effect of vaccines can include, for example, induction of cell mediated immunity or alteration of the response of the T cell to its antigen.

“immunologically effective” refers to an amount of antigen and antigen presenting cells loaded with one or more heat-shocked and/or killed tumor cells that elicit a change in the immune response to prevent or treat a cancer. The amount of antigen-loaded and/or antigen-loaded APCs inserted or reinserted into the patient will vary between individuals depending on many factors. For example, different doses may be required for an effective immune response in a human with a solid tumor or a metastatic tumor.

Inhibiting cancer progression as contemplated herein is accomplished in a variety of ways, e.g., by one or more of the following: direct cytolysis of tumor cells, direct induction of tumor cell apoptosis, induction of tumor cell cytolysis through stimulation of intrinsic host antitumor responses, induction of tumor cell apoptosis through stimulation of intrinsic host antitumor responses, inhibition of tumor cell metastasis, inhibition of tumor cell proliferation, and induction of senescence in the tumor cell.

Exemplary tumor cells contemplated for treatment herein are selected from the group of cancers consisting of: soft tissue sarcomas, kidney, liver, intestinal, rectal, leukemias, lymphomas, and cancers of the brain, esophagus, uterine cervix, bone, lung, endometrium, bladder, breast, larynx, colon/rectum, stomach, ovary, pancreas, adrenal gland and prostate. More specifically, cell lines may be selected from a group comprising of: cell lines selected from a group comprising of: J82, RT4, ScaBER, T24, TCCSUP, 5637 Carcinoma, SK-N-MC Neuroblastoma, SK-N-SH Neuroblastoma, SW 1088 Astrocytoma, SW 1783 Astrocytoma, U-87 MG Glioblastoma, astrocytoma, grade III, U-118 MG Glioblastoma, U-138 MG Glioblastoma, U-373 MG Glioblastoma, astrocytoma, grade III, Y79 Retinoblastoma, BT-20 Carcinoma, breast, BT-474 Ductal carcinoma, breast, MCF7 Breast adenocarcinoma, pleural effusion, MDA-MB-134-V Breast, ductal carcinoma, pleural I effusion, MDA-MD-157 Breast medulla, carcinoma, pleural effusion, MDA-MB-175-VII Breast, ductal carcinoma, pleural Effusion, MDA-MB-361 Adenocarcinoma, breast, metastasis to brain, SK-BR-3 Adenocarcinoma, breast, malignant pleural effusion, C-33 A Carcinoma, cervix, HT-3 Carcinoma, cervix, metastasis to lymph node ME-180 Epidermoid carcinoma, cervix, metastasis to omentum, MEL-175 Melanoma, MEL-290 Melanoma, HLA-A*0201 Melanoma cells, MS751 Epidermoid carcinoma, cervix, metastasis to lymph Node, SiHa Squamous carcinoma, cervix, JEG-3 Choriocarcinoma, Caco-2 Adenocarcinoma, colon HT-29 Adenocarcinoma, colon, moderately well-differentiated grade H, SK-CO-1 Adenocarcinoma, colon, ascites, HuTu 80 Adenocarcinoma, duodenum, A-253 Epidermoid carcinoma, submaxillary gland FaDu Squamous cell carcinoma, pharynx, A-498 Carcinoma, kidney, A-704 Adenocarcinoma, kidney Caki-1 Clear cell carcinoma, consistent with renal primary, metastasis to skin, Caki-2 Clear cell carcinoma, consistent with renal primary, SK-NEP-1 Wilms' tumor, pleural effusion, SW 839 Adenocarcinoma, kidney, SK-HEP-1 Adenocarcinoma, liver, ascites, A-427 Carcinoma, lung Calu-1 Epidermoid carcinoma grade HI, lung, metastasis to pleura, Calu-3 Adenocarcinoma, lung, pleural effusion, Calu-6 Anaplastic carcinoma, probably lung, SK-LU-1 Adenocarcinoma, lung consistent with poorly differentiated, grade HI, SK-MES-1 Squamous carcinoma, lung, pleural effusion, SW 900 Squamous cell carcinoma, lung, EB1 Burkitt lymphoma, upper maxilia, EB2 Burkitt lymphoma, ovary P3HR-1 Burkitt lymphoma, ascites, HT-144 Malignant melanoma, metastasis to subcutaneous tissue Ma!me-3M Malignant melanoma, metastasis to lung, RPMI-7951 Malignant melanoma, metastasis to lymph node, SK-MEL-1 Malignant melanoma, metastasis to lymphatic system, SK-MEL-2 Malignant melanoma, metastasis to skin of thigh, SK-MEL-3 Malignant melanoma, metastasis to lymph node SK-MEL-5 Malignant melanoma, metastasis to axillary node, SK-MEL-24 Malignant melanoma, metastasis to node, SK-MEL-28 Malignant melanoma, SK-MEL-31 Malignant melanoma, Caov-3 Adenocarcinoma, ovary, consistent with primary, Caov-4 Adenocarcinoma, ovary, metastasis to subserosa of fallopian tube, SK-OV-3 Adenocarcinoma, ovary, malignant ascites, SW 626 Adenocarcinoma, ovary, Capan-1 Adenocarcinoma, pancreas, metastasis to liver, Capan-2 Adenocarcinoma, pancreas, DU 145 Carcinoma, prostate, metastasis to brain, A-204 Rhabdomyosarcoma, Saos-2 Osteogenic sarcoma, primary, SK-ES-1 Anaplastic osteosarcoma versus Swing sarcoma, SK-LNS-1 Leiomyosarcoma, vulva, primary, SW 684 Fibrosarcoma, SW 872 Liposarcoma SW 982 Axilla synovial sarcoma, SW 1353 Chondrosarcoma, humerus, U-2 OS Osteogenic sarcoma, bone primary, Malme-3 Skin fibroblast, KATO III Gastric carcinoma, Cate-1B Embryonal carcinoma, testis, metastasis to lymph node, Tera-1 Embryonal carcinoma, Tera-2 Embryonal carcinoma, SW579 Thyroid carcinoma, AN3 CA Endometrial adenocarcinoma, metastatic, HEC-1-A Endometrial adenocarcinoma HEC-1-B Endometrial adenocarcinoma, SK-UT-1 Uterine, mixed mesodermal tumor, consistent with leiomyosarcomagrade III, SK-UT-1B Uterine, mixed mesodermal tumor, Sk-Me128 Melanoma SW 954 Squamous cell carcinoma, vulva, SW 962 Carcinoma, vulva, lymph node metastasis, NCI-H69 Small cell carcinoma, lung, NCI-H128 Small cell carcinoma, lung, BT-483 Ductal carcinoma, breast BT-549 Ductal carcinoma, breast, DU4475 Metastatic cutaneous nodule, breast carcinoma HBL-100 Breast, Hs 578Bst Breast, Hs 578T Ductal carcinoma, breast, MDA-MB-330 Carcinoma, breast MDA-MB-415 Adenocarcinoma, breast, MDA-MB-435s Ductal carcinoma, breast, MDA-MB-436 Adenocarcinoma, breast, MDA-MB-453 Carcinoma, breast, MDA-MB-468 Adenocarcinoma, breast T-47D Ductal carcinoma, breast, pleural effusion, Hs 766T Carcinoma, pancreas, metastatic to lymph node, Hs 746T Carcinoma, stomach, metastatic to left leg, Hs 695T Amelanotic melanoma, metastatic to lymph node, Hs 683 Glioma, Hs 294T Melanoma, metastatic to lymph node, Hs 602 Lymphoma, cervical JAR Choriocarcinoma, placenta, Hs 445 Lymphoid, Hodgkin's disease, Hs 700T Adenocarcinoma, metastatic to pelvis, H4 Neuroglioma, brain, Hs 696 Adenocarcinoma primary, unknown, metastatic to bone-sacrum, Hs 913T Fibrosarcoma, metastatic to lung, Hs 729 Rhabdomyosarcoma, left leg, FHs 738Lu Lung, normal fetus, FHs 173We Whole embryo, normal, FHs 738B1 Bladder, normal fetus NIH:OVCAR-3 Ovary, adenocarcinoma, Hs 67 Thymus, normal, RD-ES Ewing's sarcoma ChaGo K-1 Bronchogenic carcinoma, subcutaneous, metastasis, human, WERI-Rb-1 Retinoblastoma NCI-H446 Small cell carcinoma, lung, NCI-H209 Small cell carcinoma, lung, NCI-H146 Small cell carcinoma, lung, NCI-H441 Papillary adenocarcinoma, lung, NCI-H82 Small cell carcinoma, lung H9 T-cell lymphoma, NCI-H460 Large cell carcinoma, lung, NCI-H596 Adenosquamous carcinoma, lung NCI-H676B Adenocarcinoma, lung, NCI-H345 Small cell carcinoma, lung, NCI-H820 Papillary adenocarcinoma, lung, NCI-H520 Squamous cell carcinoma, lung, NCI-H661 Large cell carcinoma, lung NCI-H510A Small cell carcinoma, extra-pulmonary origin, metastatic D283 Med Medulloblastoma Daoy Medulloblastoma, D341 Med Medulloblastoma, AML-193 Acute monocyte leukemia MV4-11 Leukemia biphenotype.

“cancer cell antigen” refers to cells that have been stresses and killed in accordance with the present invention. Briefly, the cancer cells may be treated or stressed such that the cancer cell increases the expression of heat-shock proteins, such as HSP70, HSP60 and GP96, which are a class of proteins that are known to act as molecular chaperones for proteins that are or may be degraded. Generally, these heat-shock proteins will stabilize internal cancer cell antigens such that the cancer cells may include more highly immunogenic cancer cell-specific antigens.

“contacted” and “exposed”, when applied to an antigen and APC, are used herein to describe the process by which an antigen is placed in direct juxtaposition with the APC. To achieve antigen presentation by the APC, the antigen is provided in an amount effective to “prime” the APCs to express antigen-loaded MHC class I and/or class II antigens on the cell surface.

“therapeutically effective amount” refers to the amount of antigen-loaded APCs that, when administered to an animal in combination, is effective to kill cancer cells within the animal. The methods and compositions of the present invention are equally suitable for killing a cancer cell or cells both in vitro and in vivo. When the cells to be killed are located within an animal, the present invention may be used in conjunction or as part of a course of treatment that may also include one or more anti-neoplastic agent, e.g., chemical, irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like. The skilled artisan will recognize that the present invention may be used in conjunction with therapeutically effective amount of pharmaceutical composition such a DNA damaging compound, such as, Adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, cisplatin and the like. However, the present invention includes live cells that are going to activate other immune cells that may be affected by the DNA damaging agent. As such, any chemical and/or other course of treatment will generally be timed to maximize the adaptive immune response while at the same time aiding to kill as many cancer cells as possible.

“antigen-loaded dendritic cells,” “antigen-pulsed dendritic cells” and the like refer to DCs that have been contacted with an antigen, in this case, cancer cells that have been heat-shocked. Often, dendritic cells require a few hours, or up to a day, to process the antigen for presentation to naive and memory T-cells. It may be desirable to pulse the DC with antigen again after a day or two in order to enhance the uptake and processing of the antigen and/or provide one or more cytokines that will change the level of maturing of the DC. Once a DC has engulfed the antigen (e.g., pre-processed heat-shocked and/or killed cancer cells), it is termed an “antigen-primed DC”. Antigen-priming can be seen in DCs by immunostaining with, e.g., an antibody to the specific cancer cells used for pulsing. An antigen-loaded or pulsed DC population may be washed, concentrated, and infused directly into the patient as a type of vaccine or treatment against the pathogen or tumor cells from which the antigen originated. Generally, antigen-loaded DC are expected to interact with naive and/or memory T-lymphocytes in vivo, thus causing them to recognize and destroy cells displaying the antigen on their surfaces. In one embodiment, the antigen-loaded DC may even interact with T cells in vitro prior to reintroduction into a patient. The skilled artisan will know how to optimize the number of antigen-loaded DC per infusion, the number and the timing of infusions. For example, it will be common to infuse a patient with 1-2 million antigen-pulsed cells per infusion, but fewer cells may also induce the desired immune response.

The antigen-loaded DCs may be co-cultured with T-lymphocytes to produce antigen-specific T-cells. As used herein, the term “antigen-specific T-cells” refers to T-cells that proliferate upon exposure to the antigen-loaded APCs of the present invention, as well as to develop the ability to attack cells having the specific antigen on their surfaces. Such T-cells, e.g., cytotoxic T-cells, lyse target cells by a number of methods, e.g., releasing toxic enzymes such as granzymes and perforin onto the surface of the target cells or by effecting the entrance of these lytic enzymes into the target cell interior. Generally, cytotoxic T-cells express CD8 on their cell surface. T-cells that express the CD4 antigen CD4, commonly known as “helper” T-cells, can also help promote specific cytotoxic activity and may also be activated by the antigen-loaded APCs of the present invention. In certain embodiments, the cancer cells, the APCs and even the T-cells can be derived from the same donor whose MNC yielded the DC, which can be the patient or an HLA—or obtained from the individual patient that is going to be treated. Alternatively, the cancer cells, the APCs and/or the T-cells can be allogeneic.

The invention provides means of inducing an anti-cancer response in a mammal, comprising the steps of initially “priming” the mammal by administering an agent that causes local accumulation of antigen presenting cells. Subsequently, a tumor antigen is administered in the local area where said agents causing accumulation of antigen presenting cells is administered. A time period is allowed to pass to allow for said antigen presenting cells to traffic to the lymph nodes. Subsequently a maturation signal, or a plurality of maturation signals are administered to enhance the ability of said antigen presenting cell to activate adaptive immunity. In some embodiments of the invention activators of adaptive immunity are concurrently given, as well as inhibitors of the tumor derived inhibitors are administered to derepress the immune system.

In one embodiment priming of the patient is achieved by administration of GM-CSF subcutaneously in the area in which antigen is to be injected. Various scenarios are known in the art for administration of GM-CSF prior to administration, or concurrently with administration of antigen. The practitioner of the invention is referred to the following publications for dosage regimens of GM-CSF and also of peptide antigens [67-78]. Subsequent to priming, the invention calls for administration of tumor antigen. Various tumor antigens may be utilized, in one preferred embodiment, lysed tumor cells from the same patient area utilized. Means for generation of lyzed tumor cells are well known in the art and described in the following references [79-85]. One example method for generation of tumor lysate involves obtaining frozen autologous samples which are placed in hanks buffered saline solution (HBSS) and gentamycin 50 μg/ml followed by homogenization by a glass homogenizer. After repeated freezing and thawing, particle-containing samples are selected and frozen in aliquots after radiation with 25 kGy. Quality assessment for sterility and endotoxin content is performed before freezing. Cell lysates are subsequently administered into the patient in a preferred manner subcutaneously at the local areas where DC priming was initiated. After 12-72 hours, the patient is subsequently administered with an agent capable of inducing maturation of DC. Agents useful for the practice of the invention, in a preferred embodiment include BCG and HMGB1 peptide. Other useful agents include: a) histone DNA; b) imiqimod; c) beta-glucan; d) hsp65; e) hsp90; f) HMGB-1; g) lipopolysaccharide; h) Pam3CSK4; i) Poly I: Poly C; j) Flagellin; k) MALP-2; l) Imidazoquinoline; m) Resiquimod; n) CpG oligonucleotides; o) zymosan; p) peptidoglycan; q) lipoteichoic acid; r) lipoprotein from gram-positive bacteria; s) lipoarabinomannan from mycobacteria; t) Polyadenylic-polyuridylic acid; u) monophosphoryl lipid A; v) single stranded RNA; w) double stranded RNA; x) 852A; y) rintatolimod; z) Gardiquimod; and aa) lipopolysaccharide peptides.

Culture of dendritic cells is well known in the art, for example, U.S. Pat. No. 6,936,468, issued to Robbins, et al., for the use of tolerogenic dendritic cells for enhancing tolerogenicity in a host and methods for making the same. Although the current invention aims to reduce tolerogenesis, the essential means of dendritic cell generation are disclosed in the patent. U.S. Pat. No. 6,734,014, issued to Hwu, et al., for methods and compositions for transforming dendritic cells and activating T cells. Briefly, recombinant dendritic cells are made by transforming a stem cell and differentiating the stem cell into a dendritic cell. The resulting dendritic cell is said to be an antigen presenting cell which activates T cells against MHC class I-antigen targets. Antigens for use in dendritic cell loading are taught in, e.g., U.S. Pat. No. 6,602,709, issued to Albert, et al. This patent teaches methods for use of apoptotic cells to deliver antigen to dendritic cells for induction or tolerization of T cells. The methods and compositions are said to be useful for delivering antigens to dendritic cells that are useful for inducing antigen-specific cytotoxic T lymphocytes and T helper cells. The disclosure includes assays for evaluating the activity of cytotoxic T lymphocytes. The antigens targeted to dendritic cells are apoptotic cells that may also be modified to express non-native antigens for presentation to the dendritic cells. The dendritic cells are said to be primed by the apoptotic cells (and fragments thereof) capable of processing and presenting the processed antigen and inducing cytotoxic T lymphocyte activity or may also be used in vaccine therapies. U.S. Pat. No. 6,455,299, issued to Steinman, et al., teaches methods of use for viral vectors to deliver antigen to dendritic cells. Methods and compositions are said to be useful for delivering antigens to dendritic cells, which are then useful for inducing T antigen specific cytotoxic T lymphocytes. The disclosure provides assays for evaluating the activity of cytotoxic T lymphocytes. Antigens are provided to dendritic cells using a viral vector such as influenza virus that may be modified to express non-native antigens for presentation to the dendritic cells. The dendritic cells are infected with the vector and are said to be capable of presenting the antigen and inducing cytotoxic T lymphocyte activity or may also be used as vaccines.

In one embodiment the immunization to cancer associated fibroblasts is conducted in conjunction with approaches known to inhibit cancer. These are well known in the art and include but are not limited to, alkylating agents such as ifosfamide, nimustine hydrochloride, cyclophosphamide, dacarbazine, melphalan, and ranimustine, antimetabolites such as gemcitabine hydrochloride, enocitabine, cytarabine ocfosfate, a cytarabine formulation, tegafur/uracil, a tegafur/gimeracil/oteracil potassium mixture, doxifluridine, hydroxycarbamide, fluorouracil, methotrexate, and mercaptopurine, antitumor antibiotics such as idarubicin hydrochloride, epirubicin hydrochloride, daunorubicin hydrochloride, daunorubicin citrate, doxorubicin hydrochloride, pirarubicin hydrochloride, bleomycin hydrochloride, peplomycin sulfate, mitoxantrone hydrochloride, and mitomycin C, alkaloids such as etoposide, irinotecan hydrochloride, vinorelbine tartrate, docetaxel hydrate, paclitaxel, vincristine sulfate, vindesine sulfate, and vinblastine sulfate, hormone therapy agents such as anastrozole, tamoxifen citrate, toremifene citrate, bicalutamide, flutamide, and estramustine phosphate, platinum complexes such as carboplatin, cisplatin, and nedaplatin, angiogenesis inhibitors such as thalidomide, neovastat, and bevacizumab, L-asparaginase etc., drugs inhibiting the activity or production of the above bioactive substances, such as, for example, antibodies and antibody fragments that neutralize the above bioactive substances, and substances that suppress expression of the above bioactive substances, such as an siRNA, a ribozyme, an antisense nucleic acid (including RNA, DNA, PNA, and a composite thereof), substances that have a dominant negative effect such as a dominant negative mutant, vectors expressing same, cell activity inhibitors such as a sodium channel inhibitor, cell-growth inhibitors, and apoptosis inducers such as compound 861 and gliotoxin. Furthermore, the ‘drug controlling the activity or growth of a cancer cell’ in the present invention may be any drug that directly or indirectly promotes the physical, chemical, and/or physiological actions, etc. of a cancer cell directly or indirectly related to suppressing the onset, progression, and/or recurrence of a cancer. Among the above-mentioned drugs, an anticancer agent is particularly preferable from the viewpoint of therapeutic.

It is known in the art that exosomes secreted from tumors are critical to tumor immune suppression. One of the teachings of the current invention is that exosomes from tumors may be used to reprogram healthy fibroblasts to take a “tumor associated fibroblast” phenotype, which then allows the reprogrammed fibroblast to be utilized as a source of antigen in a tumor vaccine. Microvesicles secreted by tumor cells have been known since the early 1980s, originally described by Dr Doug Taylor [86]. They were estimated to be between 50-200 nanometers in diameter and associated with a variety of immune inhibitory effects. Specifically, it was demonstrated that such microvesicles could not only induce T cell apoptosis, but also block various aspects of T cell signaling, proliferation, cytokine production, and cytotoxicity [87-89]. Although much interest arose in the biology of microvesicles, little therapeutic applications developed since they were uncharacterized at a molecular level. Research occurring independently identified another type of microvesicular-like structures, which were termed “exosomes”. Originally defined as small 80-200 nanometers in diameter, exosomes were observed initially in maturing reticulocytes [90, 91]. Subsequently it was discovered that exosomes are a potent method of dendritic cell communication with other antigen presenting cells. Exosomes secreted by dendritic cells were observed to contain extremely high levels of MHC I, MHC H, costimulatory molecules, and various adhesion molecules [92]. In addition, dendritic cell exosomes contain antigens that said dendritic cell had previously engulfed [93]. The ability of exosomes to act as “mini-antigen presenting cells” has stimulated cancer researchers to pulse dendritic cells with tumor antigens, collect exosomes secreted by the tumor antigen-pulsed dendritic cell, and use these exosomes for immunotherapy. Such exosomes were seen to be capable of eradicating established tumors when administered in various murine models [94, 95]. The ability of dendritic exosomes to potently prime the immune system brought about the question if exosomes may also possess a tolerance inducing or immune suppressive role. Since it is established that the exosome has a high concentration of tumor antigens, the question arose if whether exosomes may induce an abortive T cell activation process leading to anergy [96]. Specifically, it is known that numerous tumor cells, and exosomes derived thereof express the T cell apoptosis-inducing molecule Fas ligand [96-98]. Fas ligand is an integral type H membrane protein belonging to the TNF family whose expression is observed in a variety of tissues and cells such as activated lymphocytes and the anterior chamber in the eye. Fas ligand induces apoptotic cell death in various types of cells target cells via its corresponding receptor, CD95/APO1. Fas ligand not only plays important roles in the homeostasis of activated lymphocytes, but it has also been implicated in establishing immune-privileged status in the testis and eye, as well as a mechanisms by which tumors escape immune mediated killing. Accordingly, given the expression of Fas ligand on a variety of tumors, we and others have sought, and successful demonstrated that Fas ligand is expressed on exosomes secreted by tumor cells [96]. Due to the ability of exosomes to mediate a variety of immunological signals, the model system was proposed that at the beginning of the neoplastic process, tumor secreted exosomes selectively induce antigen-specific T cell apoptosis, through activating the T cell receptor, which in turn upregulates expression of Fas on the T cell, subsequently, the Fas ligand molecule on the exosome induces apoptosis. This process may be occurring by a direct interaction between the tumor exosome and the T cell, or it may be occurring indirectly by tumor exosomes binding dendritic cells, then subsequently when T cells bind dendritic cells in lymphatic areas, the exosome actually is bound by the dendritic cell and uses dendritic cell adhesion/costimulatory molecules to form a stable interaction with the T cell and induce apoptosis. In the context of more advanced cancer patients, where exosomes reach higher concentrations systemically, the induction of T cell apoptosis occurs in an antigen-nonspecific, but Fas ligand, MHC I-dependent manner. The recent recognition that tumor secreted exosomes are identical to the tumor secreted microvesicles described in the 1980s [99], has stimulated a wide variety of research into the immune suppressive ability of said microvesicles. Specifically, immune suppressive microvesicles were identified not only in cancer patients [88, 100], but also in pregnancy [101-103], transplant tolerance [104, 105], and oral tolerance [106, 107] situations. Accordingly, one ideal method of stimulating the immune response of a cancer patient would be the removal of microvesicles from circulation through the use of an extracorporeal approach.

In one embodiment of the invention exosomes from tumors are concentrated and used to convert mesenchymal stem cells into tumor associated fibroblasts, said tumor exosomes are obtained from conditioned medium of cultured tumor cell lines, a descendent thereof or a cell line derived therefrom in a cell culture medium and isolating the cell culture medium. In general, we describe a method for separating a particle from other entities in a sample as a means of concentrated said tumor exosomes. The other entities may comprise things which are not of interest, and from which separation of the particle is desired. We refer to these for convenience as “contaminants”. The particle may comprise a particle from a tumor cell, such as secreted by a tumor cell. The particle may comprise a vesicle, or microvesicle, or an exosome. For example, we describe a method which comprises loading a composition comprising tumor or tumor stem cell particles onto an ion exchange resin. The ion exchange resin may comprise an anion exchange resin. The ion exchange resin may be in the form of a spin column. The composition may be loaded with an equilibration buffer. The ion exchange resin may be washed with a wash buffer. The particles may be eluted by a salt gradient. According to the methods and compositions described here, ion exchange chromatography may be used to separate and/or purify exosomes from mesenchymal stem cells. The separation may be in a small (analytical) or large (preparative) scale.

Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on their charge. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. Ion-exchange chromatography retains analyte molecules on the column based on coulombic (ionic) interactions. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. In the present example, anion exchange chromatography may be employed to purify or separate mesenchymal stem cell exosomes. Anion exchange chromatography retains anions using positively charged functional group on the resin, which binds to negatively charged exosomes from mesenchymal stem cells. In the methods described here using ion exchange chromatography, a sample comprising tumor or tumor stem cell particles (such as exosomes) is introduced, either manually or with an autosampler, into a sample loop of known volume. A buffered aqueous solution known as the mobile phase carries the sample from the loop onto a column that contains some form of stationary phase material. To optimize binding of all charged molecules, the mobile phase is generally a low to medium conductivity (i.e., low to medium salt concentration) solution. The adsorption of the charged ionic groups in the sample molecule and in the functional ligand on the support. The strength of the interaction is determined by the number and location of the charges on the molecule and on the functional group. By increasing the salt concentration (generally by using a linear salt gradient) the molecules with the weakest ionic interactions start to elute from the column first. Molecules that have a stronger ionic interaction require a higher salt concentration and elute later in the gradient. The binding capacities of ion exchange resins are generally quite high. This is of major importance in process scale chromatography, but is not critical for analytical scale separations. The stationary phase material may comprise a resin or gel matrix consisting of for example agarose or cellulose beads with covalently bonded charged functional groups. The target analytes (anions in the case of exosomes from mesenchymal stem cells) are retained on the stationary phase. The pH of the mobile phase buffer must be between the pl (isoelectric point) or pKa (acid dissociation constant) of the charged particle and the pKa of the charged group on the solid support. For example, in anion exchange chromatography a molecule with a pl of 6.8 may be run in a mobile phase buffer at pH 8.0 when the pKa of the solid support is 10.3. The composition comprising tumor or tumor stem cell particles may be applied to the ion exchange spin column at an alkaline pH. The pH may be pH 7.4 or higher, pH 7.5 or higher, pH 7.6 or higher, pH 7.7 or higher, pH 7.8 or higher, pH 7.9 or higher, pH 8.0 or higher, pH 8.1 or higher, pH 8.2 or higher, pH 8.3 or higher, pH 8.4 or higher, pH 8.5 or higher, pH 8.6 or higher, pH 8.7 or higher, pH 8.8 or higher, pH 8.9 or higher, pH 9.0 or higher, pH 9.1 or higher, pH 9.2 or higher, pH 9.3 or higher, pH 9.4 or higher, pH 9.5 or higher, pH 9.6 or higher, pH 9.7 or higher, pH 9.8 or higher, pH 9.9 or higher or pH 10.0 or higher. For example, the pH may be about pH 8.8.

Bound exosomes from tumor or tumor stem cells may be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase. The bound exosomes may be eluted by applying a gradient of linearly increasing salt concentration. Alternatively, a step gradient may be employed. This requires less complicated equipment and can be very effective to elute different fractions. For example, bound tumor or tumor stem cell exosomes may be displaced by the addition of negatively charged ions such as chloride ions. Thus, the bound tumor or tumor stem cell particles may be eluted at a high salt concentration. The salt may comprise any suitable chloride salt. The salt may comprise for example sodium chloride, i.e., NaCl. The salt concentration may be 500 μM or more, 1 mM or more, 2 mM or more, 4 mM or more, 8 mM or more, 16 mM or more, 32 mM or more, 62.5 mM or more, 125 mM or more, 250 mM or more, 500 mM or more, 1M or more or 2M NaCl or more.

Changes in pH may also be used to affect a separation. In anion exchange chromatography, lowering the pH of the mobile phase buffer will cause the particle to become more protonated and hence more positively (and less negatively) charged. The result is that the protein no longer can form a ionic interaction with the positively charged solid support which causes the molecule to elute from the column. Analytes of interest may be detected by any suitable means, typically by conductivity or UVNisible light absorbance. Alternatively or in addition, polypeptides diagnostic of the analyte of interest, such as tumor exosomes or tumor stem cell exosomes may be detected by, e.g., immunochemistry. Examples of antigens that are found on tumor exosomes include the alpha subunit of the 20S proteasome. A further example is CD9. Thus, the methods described here may include detection of either the alpha subunit of the 20S proteasome, or CD9, or both, in eluted fractions as a means to detect fractions comprising tumor or tumor stem cell particles. The alpha subunit of the 20S proteasome or CD9, or both, may be detected by any suitable means. The alpha subunit of the 20S proteasome may for example be detected by an anti-20S proteasome antibody that recognises the alpha subunits. The CD9 may be detected by an anti-CD9 antibody, or both.

It is known in the art that cancer depresses the immune system. For the practice of the invention it may be important in certain cases to “derepress” the immune system. In order to guide one of skill in the art in the practice of the invention, we will overview some of the mechanisms by which cancer suppresses the immune system. The development of such a successful immune responses to cancer is hindered by numerous factors, including primarily, that ability of the tumor to cause suppression of productive host immune system to cancer. The interaction between the tumor and the immune system can be likened to the situation of pregnancy, in which a semi-allogeneic graft (the fetus) rapidly develops in an immune competent host without rejection. The ability of the fetus to evade the immune response of the mother is not due to anatomical barriers, since maternal immune cells have been demonstrated to cross the placenta and actually enter the fetus [108]. What seems to occur in pregnancy is similar to the cancer patient in that there occurs a selective depletion of immune components, while other immunological parameters are left intact. Both in pregnancy and cancer a specific depletion of certain T cells occurs via numerous common mechanisms such as FasL [109-111]. Before elaborating on specific mechanisms by which FasL kills immune system cells, we will first overview some of the historical work that led to the notion that cancer suppresses the immune system. Experiments in the 1970s demonstrated the existence of immunological “blocking factors”, then-unidentified components of plasma found in cancer patients and pregnant women that antigen-specifically inhibited lymphocyte responses. Some of this early work involved culturing autologous lymphocytes with autologous tumor cells in the presence of third party healthy serum. This culture resulted in an inhibition of growth of the autologous tumor as a result of the lymphocytes. Third party lymphocytes did not inhibit the growth of the tumor. Interestingly when autologous serum (ie from the cancer patient) was added to the cultures, the lymphocyte-mediated inhibition of tumor growth was not observed. These experiments gave rise to the concept of antigen-specific “blocking factors” found in the body of cancer patients that incapacitate successful tumor immunity [112-114]. This work stimulated the more recent demonstration of tumor-suppression of immune function in experiments showing that T cell function is suppressed in terms of inability to secrete interferon gamma due to a cleavage of the critical T cell receptor transduction component, the TCR-zeta chain [115]. Originally, zeta chain cleavage was identified in T cells prone to undergo apoptosis [116]. Although a wide variety of explanations have been put forth for the cleavage of the zeta chain, one particular cause was postulated to be tumor-secreted microvesicles [100]. Since the immune suppressive effects of cancer are systemic, the ability of microvesicles secreted by tumor cells to specifically induce T cell modulation through circulating through-out the body has attracted considerable attention. While there are several known mechanisms of cancer to suppress the immune system that do not use microvesicles, their sheer number in the cancer patient, their ability to systemically influence numerous immune parameters, as well as the fact that administration of cancer microvesicles stimulates cancer progression, all point to their important role in cancer evasions of the immune response.

Exosomes from said tumors are cultured at concentrations with fibroblasts, in one preferred embodiment placental fibroblasts are utilized together with exosomes from cancer cell lines. At a preferred concentration approximately 0.2 ug/ml of exosomes are cultured with placentally derived fibroblast cells. Expression of CD248 is utilized as a marker of transforming fibroblasts into cells resembling tumor associated fibroblast. Said fibroblasts may be assessed for immune suppressive properties, angiogenic properties or growth promoting properties. Said fibroblasts, in a preferred embodiment are irradiated with approximately 10 Gy and subsequently administered with the patient at a concentration sufficient to elicit an immune response. In one embodiment an optimized HMGB1 peptide is coadministered. Concentration of cells administered is between one million to 100 million, once weekly for 3 months. 

1. A method of inducing an anticancer response comprising the steps of: a) obtaining a fibroblastic cell population; b) treating said fibroblastic cell population with an agent or plurality of agents capable of inducing an antigenic state resembling that found in tumor associated fibroblasts; and c) administering to a cancer patient said treated fibroblasts under conditions capable of stimulating an immune response against said fibroblasts, with said immune response resulting in recognition and destruction of tumor associated fibroblasts.
 2. The method of claim 1, wherein said fibroblastic cells are obtained from a tissue selected from a group of tissues comprising of: a) adipose; b) placental; c) skin; d) bone marrow; and e) adipose.
 3. The method of claim 1, wherein said fibroblastic cells are treated with inflammatory agents.
 4. The method of claim 3, wherein said inflammatory agents are selected from a group comprising of: a) IL-1, b) TNF-alpha; c) IL-6, d) IL-33, and e) interferon gamma
 5. The method of claim 1 wherein said fibroblasts are treated with an agent selected from a group comprising of: a) trichostatin A; b) 5-azacytidine; c) valproic acid; and e) lithium.
 6. The method of claim 1 wherein said fibroblasts are cultured in the presence of tumor conditioned media for a time sufficient to induce properties of cancer associated fibroblasts in said fibroblasts.
 7. The method of claim 1, wherein said fibroblasts are cocultured with tumor cells.
 8. The method of claim 1, wherein said fibroblasts are transfected with a tumor associated gene or a combination of said tumor associated genes.
 9. The method of claim 8 wherein said tumor associated genes are selected from a group comprising of: a) abl; b) Af4/hrx; c) akt-2; d) alk; e) alk/npm; f) am11; g) amll/mtg8; h) bcl-2, 3, 6; i) bcr/abl; j) c-myc; k) dbl; l) dek/can; m) E2A/pbx1; n) egfr; o) enl/hrx; p) erg/TLS; q) erbB; r) erbB-2; s) ets-1; t) ews/fli-1; u) fms; v) fos; w) fps; x) gli; y) gsp; z) gsp; aa) HER2/neu; ab) hox 11; ac) hst; ad) IL-3; ae) int-2; af) jun; ag) kit; ah) KS3; ai) K-sam; aj) Lbc; ak) Ick; al) Imot Imo-2; am) L-myc; an) lyl-1; ao) lyt-10; ap) lyt-10/C alpha 1; aq) mas; ar) mdm-2; as) mll; at) mos; au) mtg8/am11; av) myb; aw) MYH11; ax) new; ay) N-myc; az) ost; ba) pax-5; bb) pbxl/E2a; bc) pim-1; bd) PRAD-1; be) raf; bf) RAR/PML; bg) Ras H, K, N; bh) rel/nrg; bi) ret; bj) rhom1, rhom2; bk) ros; bl) ski; bm) sis; bn) set/can; bo) src; bp) Tal1, tal2; bq) tan-1; br) Tiam1; bs) TSC2; and bt) trk.
 10. The method of claim 1, wherein said fibroblasts are cocultured in combination with a monocytic cell population.
 11. The method of claim 10, wherein said monocytic population is a monocytic cell line.
 12. The method of claim 10, wherein said monocytic population is cultured under conditions to endow said cells with a M2 phenotype.
 13. The method of claim 12, wherein said M2 phenotype comprises of cells possessing a higher concentration of IL-10 protein as compared to freshly derived monocytic cells from peripheral circulation.
 14. The method of claim 12, wherein said M2 phenotype comprises of cells possessing a higher concentration of arginase protein as compared to freshly derived monocytic cells from peripheral circulation.
 15. The method of claim 12, wherein said M2 phenotype comprises of cells possessing a higher concentration of indolamine 2,3 deoxygease protein as compared to freshly derived monocytic cells from peripheral circulation.
 16. The method of claim 12, wherein said monocytic cells are endowed with an M2 phenotype by pretreatment with interleukin
 4. 17. The method of claim 12, wherein said monocytic cells are endowed with an M2 phenotype by pretreatment with interleukin
 13. 18. The method of claim 12, wherein said monocytic cells are endowed with an M2 phenotype by pretreatment with interleukin
 34. 19. The method of claim 1, wherein said fibroblasts are derived from a tissue associated with proliferation in an adult.
 20. The method of claim 19, wherein said tissue comprises of subintenstinal submucosa.
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